Cell-type specificity in the biotrophic interaction of Ustilago maydis and its host plant maize

生物营养真菌麦氏黑穗病菌感染其寄主植物玉米的所有空中部分，在那里它导致形成大的植物肿瘤。这个项目建立在我们最初的假设上，即美国maydis利用器官特异性的分泌效应蛋白集在不同的宿主组织中引起肿瘤。在之前的项目阶段，我们验证了这一想法，并确定了一组器官特异性效应物，这些效应物是玉米叶片中肿瘤形成所必需的，而不是花序中肿瘤的形成。其中一种效应物See1被发现是诱导玉米叶片中DNA合成所必需的，该合成在肿瘤形成开始时被真菌重新激活。对侵染玉米叶片的细胞学分析表明，玉米叶片肿瘤是由两个不同的细胞过程形成的。束鞘细胞是增生性肿瘤细胞（hyperplastic tumor cells, HPTs）的起源，而叶肉细胞则转化为肥厚性肿瘤细胞（hypertrophic tumor cells, HTTs）。引人注目的是，See1效应物作为细胞周期再激活的重要诱导剂，特别是在束鞘细胞中。采用激光捕获显微解剖技术从HTTs和hpt以及see1缺失突变体诱导的hpt中分离RNA进行转录组分析。该方法揭示了梅氏菌效应基因的细胞型特异性基因表达。此外，它还提供了一组9个候选的肥厚相关效应物。maydis与密切相关的病原体Sporisorium reilianum的转录组比较分析显示，叶片和细胞类型特异性效应物的功能与差异转录调控特别相关。基于这些结果，本项目旨在了解梅氏菌诱导肿瘤形成的细胞类型特异性机制。在确定See1是诱导植物细胞增殖所需的效应物之后，我们现在的目标是解剖U. maydis是如何诱导肥大的。为此，我们将识别和功能特征的肥厚诱导效应(s)的美国maydis。我们将确定这些效应物的植物靶点，以了解美氏菌诱导肥厚的分子基础。最后，我们提出了一种新的方法，通过CRISPR-Cas9介导的基因替代方法，对S. reilianum中的肥大/肿瘤诱导效应物进行功能分析。我们希望这个项目能够为真菌效应物在病原体引发的肥厚诱导方面提供新的见解。最终，我们的策略旨在定义一个能够重建植物肿瘤形成的功能效应集，这将有助于我们在机制水平上理解这一令人兴奋的过程。

The biotrophic fungus Ustilago maydis infects all aerial parts of its host plant maize, where it causes the formation of large plant tumors. This project builds on our initial hypothesis that U. maydis deploys organ-specific sets of secreted effector proteins to cause tumors in the different host tissues. During the previous project phase we verified this idea and identified a set of organ-specific effectors, which are required for formation of tumors in maize leaves, but not in the inflorescences. One of these effectors, See1, was found to be required for the induction of DNA synthesis in maize leaves, which is re-activated by the fungus at the onset of tumor formation. Detailed cytological analyses of infected maize leaves revealed that U. maydis induced leaf tumors are formed by two distinct cellular processes. The bundle sheath cells are the origin of hyperplasic tumor cells (HPTs), while the mesophyll is transformed into hypertrophic tumor cells (HTTs). Strikingly, the See1 effector acts as an essential inducer of the cell cycle reactivation specifically in the bundle sheath cells. Laser-capture microdissection was used to isolate RNA for transcriptome analysis from HTTs and HPTs, as well as HPTs induced by the see1 deletion mutant. This approach revealed cell-type specific gene expression of U. maydis effector gene. Moreover, it provided a set of nine candidates for hypertrophy-related effectors. A comparative transcriptome analysis of U. maydis with the closely related pathogen Sporisorium reilianum, which cannot induce leaf tumors, revealed differential transcriptional regulation being particularly relevant for the function of leaf- and cell-type specific effectors.Based on these results, this project aims to understand the cell-type specific mechanisms underlying U. maydis induced tumor formation. After having identified See1 as an effector being required for the induction of plant cell proliferation, we now aim to dissect how U. maydis induces hypertrophy. To this end, we will identify and functionally characterize the hypertrophy-inducing effector(s) of U. maydis. We will identify the plant targets of these effector(s) to understand the molecular basis of hypertrophy induction by U. maydis. Finally, we proposed a novel approach to functionally analyze hypertrophy-/tumor-inducing effectors by a CRISPR-Cas9 mediated gene replacement approach in S. reilianum.We expect this project to provide novel insights into the pathogen-triggered induction of hypertrophy by fungal effectors. Ultimately, our strategy aims to define a functional effector-set able to reconstruct tumor formation in plants, which will help us to understand this exciting process on the mechanistic level.